Locally applied biomedical barriers and microbicides have proven ineffectual in preventing sexual transmission of HIV. HIV requires intact lipid rafts, cholesterol-rich subregions in cell membranes, for entry into cells and for budding of infectious particles. Beta-cyclodextrin (?-CD), a cyclic heptasaccharide that removes cholesterol from cell membranes and disperses lipid rafts, has been shown to block HIV infection and abolish infectivity of budding HIV particles. Cholesterol is also required by other pathogens including many viruses, some of which are associated with STDs such as HSV-2. ??-CD is non-toxic and in wide human use in food products and as a carrier for drugs. A number of studies in humans and animals have shown that ?-CD can be safely applied to mucosal surfaces. Thus properly formulated, this molecule may be a safe microbicide effective against HIV and other pathogens. The central hypothesis of this proposal is that because it efficiently depletes cholesterol from lipid membranes thereby blocking virus infection and has an extensive and well-established safety profile in humans, ?-CD is an outstanding candidate for use as topical vaginal HIV microbicide. Over the first two years of this project we have shown that ?-CD blocks HIV infection at the level of cells and virus and defined mechanisms by which the compound inactivates virions. We also showed that ?-CD effectively blocked vaginal HIV transmission in a Hu-SCID mouse model. Our studies have identified three criteria essential for the most effective HIV microbicide: inactivate every virus particle;do not disrupt the natural barriers to infection;and do not induce an inflammatory response. In continuing this project we will remain focused on mechanisms while optimizing parameters for use of ?-CD as an HIV microbicide. The specific aims are: 1. To determine the effects of formulation of??-CD on its anti-HIV-1 activity;2. To determine the effect of??-CD on vaginal epithelium in vitro and in vivo in mouse and rhesus macaque models;3. To determine if deleterious effects of ?-CD on cell membranes can be attenuated by exogenous lipid;4. To determine ?-CD is synergistic with other agents In blocking HIV infection. These studies should provide important insights regarding the development of HIV microbicides generally and ?-CD in particular. The results should provide the basis for further animal studies and pilot human studies of this novel microbicide candidate.